FASTQ for hashtag antibody in which four biological replicates from BP were pooled
Each biological replicate (single cell suspension) from the Bx group was stained with unique oligo-tagged TotalSeq Type B hashtag antibody. After washing, four biological replicates were pooled together and sorted based on live and anti-human-CD45 markers. Single-cell library preparation was carried out as per the 10× Genomics Next GEM Single Cell 3' v3,1 GEM (Part Number: 1000079) and Single Cell 3' Feature Barcode Library Kit (Part Number: 1000079 (10× Genomics, Pleasanton, CA). Cell suspensions were loaded onto a Chromium Next GEM Chip G with the reverse transcription (RT) master mix and Chromium Next GEM Single Cell 3' Gel Bead Kit v3.1 (Part Number: 1000129) gel beads, aiming for 16000 cells per channel. Following the generation of single-cell gel bead-in-emulsions (GEMs), RT was performed using a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA); 11 cycles were used for cDNA amplification. Amplified cDNA was purified using SPRIselect beads (Beckman Coulter, Lane Cove, NSW, Australia) as per the manufacturer's recommended parameters. Post-cDNA amplification reaction QC and quantification were performed on the Agilent 2100 Tapestation HSD5000 Tape. For input into the gene expression library construction, 50-80 ng of cDNA was used (25% of total cDNA volume). For input into surface protein libraries, 20-40 ng of cDNA were used (12.5% of DNA sample).  Gene expression libraries went through 12 cycles of library amplification, while surface protein libraries went through 10 cycles of library amplification. Libraries were sequenced on an Illumina NovaSeq, S4, 2x150 (750 Gb). Demultiplexing of the Illumina files and generation of FASTQ files containing the scRNA-seq data were performed using the “Cell Ranger mkfastq” function in the Cell Ranger software package (version 3.1.0), a raw data pre-processing pipeline from 10× Genomics. Alignment of scRNA-seq reads to the human reference genome (GRCh38) and transcript quantification were performed using the “Cell Ranger count” function in Cell Ranger.